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. 2020 Jun 29;12(7):1455.
doi: 10.3390/polym12071455.

Assessment of the Release of Vascular Endothelial Growth Factor from 3D-Printed Poly-ε-Caprolactone/Hydroxyapatite/Calcium Sulfate Scaffold with Enhanced Osteogenic Capacity

Cheng-Yu Chen  1 , Chien-Chang Chen  2 , Chen-Ying Wang  1   3 , Alvin Kai-Xing Lee  2   4 , Chun-Liang Yeh  1 , Chun-Pin Lin  1   5
Affiliations

Assessment of the Release of Vascular Endothelial Growth Factor from 3D-Printed Poly-ε-Caprolactone/Hydroxyapatite/Calcium Sulfate Scaffold with Enhanced Osteogenic Capacity

Cheng-Yu Chen et al. Polymers (Basel). .

Abstract

Vascular endothelial growth factor (VEGF) is one of the most crucial growth factors and an assistant for the adjustment of bone regeneration. In this study, a 3D scaffold is fabricated using the method of fused deposition modeling. Such a fabricated method allows us to fabricate scaffolds with consistent pore sizes, which could promote cellular ingrowth into scaffolds. Therefore, we drafted a plan to accelerate bone regeneration via VEGF released from the hydroxyapatite/calcium sulfate (HACS) scaffold. Herein, HACS will gradually degrade and provide a suitable environment for cell growth and differentiation. In addition, HACS scaffolds have higher mechanical properties and drug release compared with HA scaffolds. The drug release profile of the VEGF-loaded scaffolds showed that VEGF could be loaded and released in a stable manner. Furthermore, initial results showed that VEGF-loaded scaffolds could significantly enhance the proliferation of human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVEC). In addition, angiogenic- and osteogenic-related proteins were substantially increased in the HACS/VEGF group. Moreover, in vivo results revealed that HACS/VEGF improved the regeneration of the rabbit's femur bone defect, and VEGF loading improved bone tissue regeneration and remineralization after implantation for 8 weeks. All these results strongly imply that the strategy of VEGF loading onto scaffolds could be a potential candidate for future bone tissue engineering.

Keywords: 3D printing; bone regeneration; calcium sulfate; hydroxyapatite; porous scaffold; vascular endothelial growth factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The photographs of (A) different view, (B) high magnification and (C) X-ray diffraction patterns of the hydroxyapatite (HA), calcium sulfate (CS) and HACS powder and (D) the 3D printed HA, HACS and PCL scaffolds.
Figure 2
Figure 2
The degradation profile of HA and HACS scaffolds after being immersed in SBF for 6 months. Data are presented as mean ± SEM, n = 6 for each group. * p < 0.05 compared with HA.
Figure 3
Figure 3
Stress–strain curves of HA and HACS scaffolds before and after immersion in SBF for 1 month and 6 months.
Figure 4
Figure 4
Vascular endothelial growth factor (VEGF) release from HA and HACS scaffolds after immersion in the cultured medium at 37 °C for 14 days.
Figure 5
Figure 5
The proliferation of (A) human mesenchymal stem cells (hMSCs) and (B) human umbilical vein endothelial cells (HUVECs) cultured on different 3D-printed scaffolds. “*” indicates a significant difference (p < 0.05) when compared to the scaffold without VEGF. “#” indicates a significant difference (p < 0.05) when compared to HA/VEGF.
Figure 6
Figure 6
The F-actin filaments (green) and nuclei (blue) staining of hMSCs and HUVECs cultured on the scaffolds for 3 and 7 days. The scale bar is 400 µm.
Figure 7
Figure 7
(A) The von Willebrand factor (vWF) and (B) Angiopoietin-1 (Ang-1) protein expression levels of HUVECs cultured on various scaffolds for 3 and 7 days. “*” indicates a significant difference (p < 0.05) when compared to the scaffold without VEGF. “#” indicates a significant difference (p < 0.05) when compared to HA/VEGF.
Figure 8
Figure 8
(A) The ALP and (B) OC protein expression levels of hMSCs cultured on various scaffolds for various time-points. (C) Alizarin red S staining and quantification of Ca mineral deposits after culture for 14 days. Data are presented as mean ± SEM, n = 3 for each group. “*” indicates a significant difference (p < 0.05) when compared to the scaffold without VEGF. “#” indicates a significant difference (p < 0.05) when compared to HA/VEGF.
Figure 9
Figure 9
Evaluation of bone formation in vivo. (A) Micro-CT images at week 8. (B) Micro-CT-quantified histograms of (C) bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th). “*” indicate a significant difference (p < 0.05) when compared to HA.
Figure 10
Figure 10
Histological analysis of new bone regeneration around and within the scaffolds in the rabbit femoral defect model. Left: hematoxylin and eosin (HE) stain; middle: Masson’s trichrome (MT) stain; right: von Kossa (VK) stain of regenerated bone mass after 8 weeks of regeneration for in vivo experiment.
Figure 11
Figure 11
The higher magnification images of MT staining of regenerated bone mass after 8 weeks of regeneration for in vivo experiment. Red arrowheads indicate the blood vessels.
See this image and copyright information in PMC

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